Linoleic acid improves PIEZO2 dysfunction in a mouse model of Angelman Syndrome

Angelman syndrome (AS) is a neurogenetic disorder characterized by intellectual disability and atypical behaviors. AS results from loss of expression of the E3 ubiquitin-protein ligase UBE3A from the maternal allele in neurons. Individuals with AS display impaired coordination, poor balance, and gait ataxia. PIEZO2 is a mechanosensitive ion channel essential for coordination and balance. Here, we report that PIEZO2 activity is reduced in Ube3a deficient male and female mouse sensory neurons, a human Merkel cell carcinoma cell line and female human iPSC-derived sensory neurons with UBE3A knock-down, and de-identified stem cell-derived neurons from individuals with AS. We find that loss of UBE3A decreases actin filaments and reduces PIEZO2 expression and function. A linoleic acid (LA)-enriched diet increases PIEZO2 activity, mechano-excitability, and improves gait in male AS mice. Finally, LA supplementation increases PIEZO2 function in stem cell-derived neurons from individuals with AS. We propose a mechanism whereby loss of UBE3A expression reduces PIEZO2 function and identified a fatty acid that enhances channel activity and ameliorates AS-associated mechano-sensory deficits.


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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. For electrophysiological experiments we excluded from the analyses (exclusion criteria were pre-established): Recordings with leak currents > 200pA, with access resistance >10M!, and cells with giga-seals that did not withstand at least five consecutive steps of mechanical stimulation were excluded from analyses. For human DPSC-derived neurons, cells with giga-seals notwithstanding at least four consecutive steps of mechanical stimulation were excluded. For CatWalk experiments (exclusion criteria were pre-established): runs shorter than 0.5 seconds, longer than 5 seconds, and with a speed variation higher than 60% across the run were excluded. For Tail-Clip: A cut-off latency of 60 s was imposed to avoid tissue damage. Animals that did not respond at this time were excluded. For Rotarod: Mice that fell 3 times before 10 seconds or performing 2 full passive rotations clinging are excluded.
All attempts at replication were successful. Experiments were performed at least 3 times in different days with different/independent preparations.
The experiments were not randomized. For electrophysiological experiments, daily measurements included the control and treated samples, and only cells with good surface attachment were chosen (e.g., healthy morphology and visible pseudopodia). Transfected cells had a Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

Antibodies
Antibodies used fluorescent transfection marker, and cells that endogenously express PIEZO2 (neurons and MCC13 cells) were chosen indiscriminately. Cell size was taken into account by normalizing each cell's current magnitude (pA) by their capacitance (pF; which is proportional to the cell size).
For behavioral experiments, we measured the behavior of all individuals corresponding to their genotype and treatment. Mice were tested in no particular order.
For electrophysiology, the investigator was blind to genotype and treatment. For behavioral assays, the investigator was blind when possible; however, diet smell and consistency can be easily identified. Moreover, the fur of animals on oily-based diets (i.e., linoleic acid) looks shinier than when fed with other diets.
No commonly misidentified cell lines were used on this study.
Mice/Strains: C57BL/6 (Stock No. 000664); C57BL/6 Ubeatm1Alb (B6 AS; Stock No. 016590) for maternal and paternal transmission. Age of animals use or experimentation was from 6 weeks to 5 months. Mice were housed with a 12 h light/dark cycle at 21°C with 40-60% humidity, with food and water ad libitum